Wound repair and regeneration are two separable biological processes by which organisms heal wounds. As a model of regeneration in mammals, ear hole closure, as seen in rabbits, in the inbred mouse strains MRL/MpJ and LG/J and other mutant mouse strains shows multiple similarities to limb regeneration in amphibians including the replacement of cartilage and the lack of scarring. Other less well known classical regenerative phenotypes are also seen in the MRL ear injury model include rapid re-epithelialization, enhanced tissue remodeling, basement membrane breakdown, blastema growth and re-differentiation not seen during wound repair. Inflammation is now considered a factor in regenerative processes and its role in ear hole closure has been further demonstrated in genetically selected, pro-inflammatory AIR (acute inflammatory reactivity) mice which have the ability to close ear holes. Another aspect of adult MRL mice is the use of aerobic glycolysis for normal metabolism, which may contribute to the regenerative response. This metabolic state contributes to inflammation, with glycolysis playing an important role in migration and activity of inflammatory cells. The molecule hypoxia inducible factor (HIF1a) is a central node in all of these and could potentially be a main actor in the MRL ear hole closure response.
HIF1a is an oxygen-regulated protein which functions as part of a heterodimeric complex formed with HIF1b in the nucleus and binds to DNA at specific promoter or enhancer sites (i.e., HREs or hypoxia response elements), thereby regulating the transcription of over 100 gene products. These include molecules of interest in regenerative processes involved in 1) angiogenesis through the induction of VEGF, VEGFR-1, and PDGF and erythropoietin (EPO); 2) tissue remodeling with the induction of uPAR, MMP2 and 9 and TIMPs; and 3) glycolytic metabolism with the induction of lactate dehydrogenase (LDH) which converts pyruvate into lactate and of pyruvate dehydrogenase kinase (PDK) which blocks pyruvate's entry into the TCA cycle. HIF1a protein is generally short-lived in the cytoplasm because under normoxic conditions, it is continually being hydroxylated by prolyl hydroxylases (PHDs), then bound by the von Hippel-Lindau tumor suppressor protein (pVHL) and the more recently identified SAG/ROC/RBX2 E3 ubiquitin ligase complex which targets the molecule for proteolysis. (M. Tan, Q. Gu, H. He, D. Pamarthy, G. L. Semenza, Y. Sun, SAG/ROC2/RBX2 is a HIF-1 target gene that promotes HIF-1 alpha ubiquitination and degradation. Oncogene 27, 1404-1411 (2008).) In low oxygen, hydroxylation is inhibited and HIF1a protein survives and is translocated to the nucleus where it binds HIF1b and can now function as a transcription factor, binding to the appropriate DNA elements or HRE. (R. H. Wenger, D. P. Stiehl, G. Camenisch, Integration of oxygen signaling at the consensus HRE. Sci. STKE 2005, re12 (2005).)
The stabilization of HIF1a protein has been accomplished through the inhibition of PHDs, molecules actively involved in collagen secretion and crosslinking PHDs control collagen deposition in fibrosis, response to ischemia, and wound repair. (See, e.g., X. J. Zhang, L. X. Liu, X. F. Wei, Y. S. Tan, L. Tong, R. Chang, G. Marti, M. Reinblatt, J. W. Harmon, G. L. Semenza, Importance of hypoxia-inducible factor 1 alpha in the healing of burn wounds in murine model. Wound Repair Regen. 17, A87-A87 (2009); T. J. Franklin, W. P. Morris, P. N. Edwards, M. S. Large, R. Stephenson, Inhibition of prolyl 4-hydroxylase in vitro and in vivo by members of a novel series of phenanthrolinones. Biochem. J. 353, 333-338 (2001); I. Kim, J. E. Mogford, C. Witschi, M. Nafissi, T. A. Mustoe, Inhibition of prolyl 4-hydroxylase reduces scar hypertrophy in a rabbit model of cutaneous scarring. Wound Repair Regen. 11, 368-372 (2003).) Considering the impact of scar formation on regeneration, inhibition of PHDs could have a two-fold effect; the up-regulation of HIF1a and the down-regulation of scarring. In a chronic diabetic wound model, the use of PHD-inhibiting compounds applied locally to a wound can accelerate wound repair in the presence of increased vascularity and granulation tissue. (I. R. Botusan, V. G. Sunkari, O. Savu, A. I. Catrina, J. Grunler, S. Lindberg, T. Pereira, S. Yla-Herttuala, L. Poellinger, K. Brismar, S. B. Catrina, Stabilization of HIF-1 alpha is critical to improve wound healing in diabetic mice. Proc. Natl. Acad. Sci. U.S.A. 105, 19426-19431 (2008).)
However, the effectiveness of various PHD inhibitor compounds can be compromised by low solubility under physiological conditions, inefficient routes of administration and/or untimely delivery of therapeutic dose levels. There remains an on-going concern in the art to provide a delivery system and methodology to better utilize the benefits and advantages available through such inhibitor compounds.